Data are expressed because the mean SD. is significantly less immunogenic in three strains of mice, yet retains full cytotoxic and anti-tumor activities. Elimination of B-cell epitopes is a promising approach to the production of less immunogenic proteins for therapeutic purposes. Keywords:antibody engineering, BL22, HA22, immunotherapy,Pseudomonasexotoxin A Immunotoxins (ITs) are hybrid proteins that are composed of a SB-649868 cancer-specific antibody attached to a bacterial or plant toxin (1). Initially ITs were made by chemically coupling toxins to whole antibodies. Now they are made using a combination of antibody and protein engineering (2,3). ITs kill cells by binding to a cell surface protein, being internalized by endocytosis and eventually reaching the cytosol, where they arrest protein synthesis by inactivating EF2 or ribosomes (4,5). Our laboratory has developed recombinant immunotoxins (RITs) in which the Fv portion of an antibody is directly fused to a 38-kDa portion of the bacterial toxinPseudomonasexotoxin A (PE). Three RITs are currently in clinical trials and all three have shown anti-tumor activity in phase 1 trials. LMB-2 [anti-Tac-(Fv)-PE38] targets CD25 expressed on many T cell malignancies and some B cell malignancies (6). BL22 [anti-CD22-(Fv)-PE38] targets CD22 expressed on most B cell malignancies (7), and SS1P anti-mesothelin-(Fv)-PE38 targets the mesothelin antigen expressed on mesotheliomas and on ovarian, lung, pancreatic, and gastric cancers (8). Because these ITs contain a portion of a bacterial protein, they can induce the formation of neutralizing antibodies, hindering their efficacy. In patients with B- and T-cell malignancies the formation of neutralizing antibodies is infrequent because of the immune-suppressed state of patients with these malignancies (6,7). However, in patients with solid tumors treated with SS1P and other ITs, antibody formation was very frequently detected 21 days after the first treatment cycle, preventing readministration of the IT (9). Previous studies have shown that the formation of antibodies to foreign proteins can be prevented by coupling the protein to high-molecular-weight polyethylene glycol (10). We have had limited success with this approach because of inactivation of the IT and only minor decreases in immunogenicity. Another approach is to SB-649868 treat patients with cyclophosphamide or fludarabine that damages the immune system (11,12). Alternative approaches are to identify and remove B-cell or T-cell epitopes (1317). We have recently used a mouse model to identify the major B-cell epitopes in the PE38 portion of RITs made by our group (18). Our approach was to immunize mice, with PE38-containing ITs, isolate monoclonal antibodies (mAbs) reacting with conformational epitopes on PE38, and use these to determine the number SB-649868 of epitopes on PE38. We Rabbit Polyclonal to ATG4A found that PE38 contains seven major conformational epitopes located in specific positions on the protein and not diffusely distributed over the entire surface of PE38. The finding that the epitopes are clustered enabled us to determine the precise location of most of the epitopes by mutating large hydrophilic amino acids on the surface of PE38 to alanine or glycine and showing that specific mAb binding to the selected epitope was abolished or greatly decreased (18). These results indicated that we might be able to reduce significantly the immunogenicity of PE38, if we combined in one IT several individual mutations that each by itself eliminated one epitope. We describe here the production of a PE38-containing IT that is significantly less immunogenic than its parental IT and does not contain new epitopes yet retains full cytotoxic activityin vitroand in mice with lymphomas. == Results == ==.
